Enzymatic immunological method for determination of antigens and antibodies

ABSTRACT

The present invention relates.Iadd., inter alia, .Iaddend.to improvements in the sandwich technique for the determination of a component of an antigen-antibody reaction in a liquid sample to be tested, utilizing as reagents (a) one component of said reaction bound to the surface of a water-insoluble, water-insuspensible, solid carrier, and (b) a component having the same immunological properties covalently linked to an enzyme. .Iadd.If and only if the component of the reagent that is water-insoluble and water-insuspensible has the same immunochemical properties as the component to be determined, is a predetermined amount of a binding partner for said first reagent (e.g., the reagent that is water-insoluble and water-insuspensible) added to the liquid sample. .Iaddend.The liquid sample is contacted and incubated with the reagent(s) to form a reaction mixture, the enzyme activity of either the liquid or solid phase of which is a measure of the presence and quantity of the component to be determined. The method is especially useful for diagnostic testing for hepatitis or rubella antibodies.

BACKGROUND OF THE INVENTION

This invention relates to a diagnostic method for the .[.direction.]..Iadd.detection .Iaddend.and determination of antigens and antibodies.More particularly, this invention relates to diagnostic methods for thedetection and determination of pathogenic disease antigens andantibodies, such as hepatitis and rubella antigens and their associatedantibodies.

A number of immunological methods have been developed for thedetermination of antigens and antibodies, including methods for thedetermination of hepatitis B surface antigen (HB₂ Ag), a component ofhepatitis B virus and its associated antibodies.

In this field it is important that whatever methods are used are assensitive and reliable as possible since an incorrect diagnosis can haveserious consequences. This is especially true of the determination ofhepatitis antigens and their associated antibodies since thetransmission of the hepatitis virus via blood donors constitutes asignificant public health risk.

Up to the present time, the radio-immunoassay (RIA) method in itsvarious forms has been the most sensitive system available. This methodhas several disadvantages, however, including the requirement of specialequipment, trained staff, the need for extra safety measures to protectagainst harmful radiation, and the short half-life span of theradioactive labelling element. The possibility of replacing theradioactive label with an enzyme label was proposed in 1968 in anarticle by L. E. M. Miles and C. N. Hales, entitled "Labelled Antibodiesand Immunological Assay Systems," Lancet, London 1968 II, page 492;Nature, Vol. 219, pages 186-189 (July 13, 1968), but no proceduraldetails were provided, the article failing to offer more than thegeneral idea, leaving it to future workers to determine the basic stepsand to perform the extensive experimentation needed to establish apractical operative enzymic immunoassay method.

The pioneering work on enzyme-immunoassay (EIA) methodology wasperformed by Schuurs and coworkers, and is disclosed in a series oftheir U.S. Pat. Nos. 3,654,090, 3,791,932, 3,850,752, 3,839,153, and3,879,262.

In the course of the further evolution of the radio-immunoassayprocedures by numerous workers, it was pointed out out in an article byE. Habermann, entitled "A new Principle for the QuantitativeDetermination of High Molecular Antigens (Junction Test), etc."published in Z. klin. Chem. u. klin. Biochem., 8th year, January, 1970,pages 51-55, that those methods based on the competition ofradio-labelled and unlabelled antigens for a limited amount ofantibodies gave better results than other methods.

The Habermann article proposed an RIA method .[.in.]. which .[.in afirst step,.]. .Iadd.involved reacting an unknown bindable substancewith an insolubilized binding partner to insolubilized the unknown. Theinsolubilized unknown was then, in turn, reacted with a radioactivelylabelled binding partner, and the quantity of insolubilizedradioactivity determined. More specifically, Habermann proposed an RIAmethod whereby .Iaddend.the antigen is adsorbed on an antibody fixedwith a covalent bond by a solid phase, e.g., celluose; a second stepconsists .[.in.]. .Iadd.of .Iaddend.fixing .[.by.]. labelled antibodies.Iadd.to .Iaddend.those antigen determinants remaining accessible. Thenon-fixed component of the antibody preparation is removed by washing.The radioactivity of the solid phase is correlated with the antigencontent of the original solution. Since the labelled and solid phaseantibodies are joined via the antigen, the author called the techniquethe "junction test." This arrangement is also known in the art as the"sandwich technique or method." The Habermann article, referred to theearlier suggestion of Miles and Hales (loc. cit.), and contained apassing suggestion that the test could be improved in sensitivity bycoupling the antibody molecule with an easily detectable enzyme, butalso offered no further suggestions, leaving it to others to developsuch an enzyme method. .Iadd.Haberman does not set forth any specificassay employing enzymes. Apart from the general statement referred toabove, Habermann merely cites a supposed suggestion by Miles et al abouthow to use enzymes--but the referenced Miles et al article contains nomention of enzymes..Iaddend.

In Schuurs et al. U.S. Pat. No. 3,791,932, there is disclosed, inExample III, .[.a rudimentary.]. .Iadd.first-generation.Iaddend.sandwich-type procedure for the determination of humanchorionic gonadotropin (HCG) and luteinizing hormone (LH) in lowconcentrations by means of an enzyme-antibody coupling component, inaccordance with which a predetermined amount of .[.HCG.]. .Iadd.anti-HCG.Iaddend. is first coupled to a solid immuno-adsorbent(m-aminobenzyloxymethyl cellulose). .[.Antibodies.]. .Iadd.PurifiedAntibodies .Iaddend.from rabbit anti-HCG serum are then coupled to anenzyme (horse radish peroxidase, HRP) .[.and the coupling product isbound to HCG cellulose.].. .Iadd.The test proceeds when an .Iaddend.HCG.[.and.]. .Iadd.or .Iaddend.LH dilution series .[.are.]. .Iadd.is.Iaddend.mixed with anti-HCG cellulose .[.to form an immunoadsorbent, towhich.]. .Iadd.immunoadsorbent forming an antigen-antibody complex. Then.Iaddend.a given amount of the antibody-enzyme coupling product isadded, and the enzyme activity of the supernatant liquid is determined.This procedure is somewhat involved and requires a number of coordinatedoperating steps.

It is an object of the present invention to develop and further improveand simplify the early version of the sandwich technique as applied tothe determination of antigens and antibodies.

.Iadd.It is an object of the present invention to use a water-insoluble,water-insuspenssible solid carrier for binding one component of anantigen-antibody reaction.

It is another object of the present invention to provide a technique forthe determination of antigens and antibodies that has sensitivity asgood as the prior art RIA methods..Iaddend.

GENERAL DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a novel,very simple and sensitive enzyme immunoassay (EIA) test systemespecially adapted for the detection and determination of a component ofan antigen-antibody reaction. The general principle of the procedure ofthe invention is that of employing as a test reagent a given amount ofone component of said reaction bound to the surface of awater-insoluble, water-insuspensible solid carrier, and as a secondreagent, a given amount of the same component covalently linked to anenzyme. The liquid sample containing the component to be determined iscontacted with these reagents. In case the component to be determinedand the component bound to the solid carrier are the same, a givenamount of a binding partner of said component is added to the liquidsample. Upon mixing the reagents and the sample, there is formed areaction mixture having a solid phase and a liquid phase. Finally theenzyme activity of the solid or liquid phase is determined, saidactivity being a measure of the presence and quantity of the componentto be determined.

The enzyme-immunoassay (EIA) method according to the present inventiondoes not possess the disadvantages of a radioimmunoassay method, and issimpler to set up and perform than the earlier EIA methods, but,surprisingly, is as sensitive as the RIA method. One of the advantagesof the EIA according to the present invention is that by using an enzymeas a labelling means the immunological reaction can be measured by acolor reaction. Either a colored substrate or a colored end-productshould participate in the enzyme-catalyzed reaction so that thedetection can take place by reading with the naked eye or by measurementof the extinction.

More specifically, the method of the present invention comprisescontacting a liquid sample, for example serum or plasma, containing anunknown amount of antigen or antibody, with a predetermined amount ofeither the .[.antigen.]. .Iadd.antibody .Iaddend.or the .[.antibody.].,.Iadd.antigen respectively, .Iaddend.which is bound to the surface of awater-insoluble, water-insuspensible, solid carrier. The liquid sampleis then incubated with this coated solid carrier for a period of 0.5 to35 hours, usually at a temperature between 4° and 50° C. Afteraspiration and washing, the foregoing solid phase is contacted with anamount of the same component covalently linked to an enzyme. If thecomponent to be determined in the liquid sample, and the componentcoupled to the solid carrier are the same, the solid phase is contactedwith a binding partner for the component to be determined.

The binding partner is employed in an insoluble form. The determinationcan take place by adding the binding partner in an insoluble form, or itcan be added in a dissolved form, and insolubilized afterwards. Thebinding partner is preferably a protein capable of binding the antigenor antibody specifically.

After an incubation period, usually from 0.5 to 25 hours, and at atemperature between 4° and 50° C., aspiration and washing, the enzymaticactivity bound to the solid phase is determined, which activity is ameasure of the pressure of the antigen or antibody component in theliquid sample tested.

Both for the detection and quantitative determination of the antigen orantibody, the foregoing reagents are employed in predetermined amounts.

The solid carrier to which one of the components is bound may be anywater-insoluble, water-insuspensible, solid carrier. Examples ofsuitable solid carriers include large beads, e.g., of polystyrene,filter paper, test tubes, and microtiter plates. The immunologicalcomponent may be bound to the solid carrier by covalent bonds or byadsorption. The advantage of the use of a solid carrier is that nocentrifugation step is needed for the separation of solid and liquidphase.

As a solid carrier, use is preferably made of a test tube of amicrotiter plate the inner walls of which are coated with theimmunological component.

The antigen or antibody enzyme conjugate consists of the immunologicalcomponent covalently linked to one or more enzyme molecules. Suchlinking can be achieved either by direct condensation or by usingexternal bridging molecules, in accordance with methods known to thoseskilled in the art.

Thus, the production of enzyme coupling products employing a covalentbond can be effected by reagents such as carbodiimides, diisocyanates,glutaric aldehyde, and bis-diazobenzidine.

The choice of the enzyme that is to form a part of the coupling productis determined by properties such as the specific binding activity (ahigh conversion rate increases the sensitivity of the test system) andthe simplicity of determination of the enzyme. The determination of anenzyme catalyzing a conversion in which colored reaction components areinvolved, is simple. Such colorimetric determinations can be automaticin a simple manner. It is also possible to employ enzymes catalyzingthose conversions in which reaction components are involved that can bedetermined spectrophotometrically or fluorimetrically. Thesedeterminations are also suitable for automation, which is an additionaladvantage.

As enzymes suitable for the method of the invention there can beemployed catalase, peroxidase, urease, glucose oxidase, alkalinephosphatase. Horse radish peroxidase is preferred.

The method according to the present invention can be used for thedetection and determination of antigens and antibodies in general,including bacteria, viruses, hormones, proteins and their associatedantibodies, but is particularly suited for the detection anddetermination of hepatitis B surface antigen and rubella and theirassociated antibodies. The invention will be illustrated with respect tothese examples but is not to be considered as limited thereto.

In order to compare the enzyme-immunoassay of the invention with thecorresponding radio-immunoassay, for the detection of HB_(S) Ag, twoHB_(S) Ag-containing sera (1 subtype ad, 1 subtype ay) were taken andwere diluted in two different normal sera, free from HB_(S) Ag andanti-HB_(S). The results from these four dilution series are indicatedin the accompanying drawing. They demonstrate that EIA and RIA haveabout the same sensitivity, but that the EIA gives a steeperdose-response curve with the serum containing subtype ay.

The enzyme immunoassay for HB_(S) Ag according to the present inventiondetected all antigen-positive samples "A," "B" and "C" of the NIHreference panel No. 2, both by eye reading and extinction measurement.This means that the assay system according to the present invention hasat least "third generation" sensitivity as defined in the FederalRegister, Vol. 39, No. 132, of July 9, 1974.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples serve to illustrate the practice of theinvention, but are not to be regarded as limiting:

EXAMPLE I Detection of Hepatitis B Surface Antigen In Human Serum orPlasma

0.1 ml test sample (human serum or plasma) is added to each well of apolystyrene microtiter plate to the inner walls of which sheepanti-(Hepatitis B surface antigen) is bound. The plate is incubated for2 hours at 37° C. The wells are emptied by aspiration. Each well iswashed three times with 0.2 ml 0.2 M TRIS(trihydroxy-methylaminomethane; 2-amino-2-hydroxymethyl-1,3-propanediol)buffer, pH 7.4 (buffer A). 0.1 ml Horse radish peroxidase coupled tosheep anti-(Hepatitis B surface antigen) in a predetermined dilution inBuffer A is added. The plate is incubated for 2 hours at 37° C. Thewells are emptied by aspiration. Each well is washed four times with 0.2ml buffer A. 0.1 ml of a substrate solution of:

0.4 mg orthophenylene diamine per ml

0.2 mg urea-peroxidase per ml

in a McIlvain buffer of pH 5.0. is added to each well. The plate isincubated for 60 minutes at room temperature. The reaction is stopped byadding 0.05 ml of 0.5 N sulphuric acid. The color is read by eye ormeasured colorimetrically at 492 nm.

A result is called positive if the ratio is ≧2.1, i.e., the averageextinction is at least 2.1 times the average extinction of six negativecontrol sera.

Result A

Titration of two Hepatitis B surface antigen-positive sera of the ay andad subtypes in enzyme immunoassay and a radio-immunoassay licensed bythe U.S. Federal Drug Administration. The positive sera were diluted intwo sera negative for Hepatitis B surface antigen and foranti-(Hepatitis B surface antigen), so that the serum concentration wasthe same in all dilutions. The curves of the ad subtype in both testsare represented by one line. (See accompanying drawing).

Result B

Reaction of the NIH Reference panel No. 2 in the abovementioned assaysystem.

    ______________________________________                                        lot No.                                                                             ratio    eye-reading                                                                              lot No.                                                                             ratio  eye-reading                            ______________________________________                                        201   >26.     +          229   1.51   -                                      202   >26.     +          230   1.72   -                                      203   0.97     -          231   1.51   -                                      204   10.33    +          232   >26.   +                                      205   >26.     +          233   1.15   -                                      206   >26.     +          234   >26.   +                                      207   1.79     -          235   >26.   +                                      208   >26.     +          236   1.56   -                                      209   >26.     +          237   1.16   -                                      210   >26.     +          238   >26.   +                                      211   1.11     -          239   >26.   +                                      212   1.25     -          240   1.39   -                                      213   >26.     +          241   1.38   -                                      214   >26.     +          242   1.23   -                                      215   2.05     -          243   >26.   +                                      216   1.32     -          244   >26.   +                                      217   >26.     +          250   >26.   +                                      218   >26.     +          251   >26.   +                                      219   >26.     +          252   >26.   +                                      220   >26.     +          253   >26.   +                                      224   1.04     -          254   >26.   +                                      225   1.16     -          255   >26.   +                                      226   >26.     +          256   >26.   +                                      227   1.36     -          260   >26.   +                                      228   >26.     +          261   >26.   +                                      ______________________________________                                    

EXAMPLE II Detection of antibodies against hepatitis B surface antigenin human serum of plasma

0.1 ml test sample (human serum or plasma) is added to each well of apolystyrene microtiter plate to the inner walls of which sheepanti-(Hepatitis B surface antigen) is bound. 0.05 ml of a predetermineddilution of hepatitis B surface antigen in 0.2 M TRIS buffer pH 7.4 isadded to each well. The plate is incubated for 2 hours at 37° C. andtreated further in the same way as in Example 1.

Results

Extinction after testing a dilution series of an anti-(hepatitis Bsurface antigen)-containing serum in antigen-and antibody-negative serumand six antigen- and antibody-negative control sera. Serum positive foranti-(Hepatitis B surface antigen)

    __________________________________________________________________________    Undiluted                                                                           1/2 1/4                                                                              1/8 1/16                                                                             1/32                                                                              1/64                                                                             1/128                                                                             1/256                                                                             1/512                                      0.050 0.051                                                                             0.049                                                                            0.062                                                                             0.060                                                                            0.058                                                                             0.168                                                                            0.200                                                                             0.203                                                                             0.198                                      __________________________________________________________________________    Negative control sera undiluted:                                              serum No.                                                                     1     2      3     4      5     6                                             __________________________________________________________________________    0.201 0.197  0.232 0.205  0.212 0.218                                         __________________________________________________________________________     A reaction is called positive is the extinction is ≦50% of the     average extinction of 6 negative control sera.

EXAMPLE III Detection of antibodies against Rubella virus in human serumor plasma

0.5 ml of test sample (human serum or plasma) is added to polystyrenetubes φ 1 cm to the inner walls of which inactivated Rubella virus iscoupled. The tubes are incubated for 1 hour at 37° C. The tubes areemptied by aspiration. Each tube is washed three times with 2 ml 0.15 Mphosphate buffer. 0.5 ml horse radish peroxidase coupled to inactivatedRubella virus in a predetermined dilution in 0.2 M TRIS buffer +0.1%triton X₁₀₀ is added. The tubes are incubated for 3 hours at 37° C. Eachtube is washed three times with 2 ml 0.2 M TRIS buffer+0.1% triton X₁₀₀.The horse radish peroxidase activity is determined by adding 0.5 mlsubstrate solution (see example I) and incubating for 60 minutes at 37°C. The reaction is stopped by adding 0.25 ml of 0.5 N sulphuric acid.The color is measured colorimetrically at 494 nm. A result is calledpositive if the ratio is ≧2.9, i.e., the average extinction is at least2.9 times the average extinction of 8 negative control sera.

Results

Extinction after testing a dilution series of ananti-(Rubella-virus)-containing serum in serum negative for Rubellavirus and anti-(rubella virus), and eight negative control sera. Serumpositive for anti-rubella-virus:

    __________________________________________________________________________    1/32                                                                              1/64                                                                              1/128                                                                             1/256                                                                             1/512                                                                             1/1024                                                                            1/2048                                                                            1/4096                                                                            1/8192                                                                            1/6384                                    >2.000                                                                            >2.000                                                                            >2.000                                                                            1.653                                                                             0.985                                                                             0.499                                                                             0.285                                                                             0.175                                                                             0.103                                                                             0.095                                     __________________________________________________________________________    Negative control sera undiluted:                                              serum No.                                                                     1    2    3    4    5    6    7    8                                          __________________________________________________________________________    0.058                                                                              0.092                                                                              0.070                                                                              0.101                                                                              0.085                                                                              0.063                                                                              0.058                                                                              0.082                                      __________________________________________________________________________

EXAMPLE IV Detection of rubella virus in humn serum or plasma

0.5 ml of test sample (human serum or plasma) is added to 0.1 ml of apredetermined solution of rabbit anti-rubella-virus. The mixture isincubated for 1 hour at 37° C. 0.5 ml of this mixture is added to apolystyrene tube φ1 cm to the inner walls of which inactivated rubellavirus is bound. The tubes are incubated for 1 hour at 37° C. and treatedfurther inthe same way as in Example III.

Results

Extinctions after testing a dilution series of a rubellavirus-containing serum in serum negative for rubella-virus andanti-rubella-virus. Serum positive for rubella virus:

    __________________________________________________________________________    1/100                                                                             1/200                                                                             1/400                                                                             1/800                                                                             1/1600                                                                            1/3200                                                                            1/6400                                                                             1/12800                                                                            1/25600                                     0.075                                                                             0.076                                                                             0.073                                                                             0.090                                                                             0.088                                                                             0.097                                                                             0.201                                                                              0.252                                                                              0.0249                                      __________________________________________________________________________    Negative control sera undiluted:                                              serum No.                                                                     1   2    3    4    5    6    7    8                                           __________________________________________________________________________    0.250                                                                             0.238                                                                              0.278                                                                              0.275                                                                              0.263                                                                              0.291                                                                              0.0258                                                                             0.261                                       __________________________________________________________________________     A reaction is called positive if the extinction is ≦50% of the     average extinction of 8 negative control sera.

For the performance of the method according to the invention, a testpack or kit of the reagents is preferably employed, chiefly composed of:

a. a given quantity of a component of the antigen-antibody reactionbound to a water-insoluble, water-insuspensible solid carrier;

b. a substance having the same immunological properties as saidcomponent, covalently linked to an enzyme;

c. the binding partner of the component to be determined if thecomponent has the same immunological properties as the component in (a).According to their nature, these reagents can be preserved byfreeze-drying or dissolved in a buffer. Thus, a specific test pack maycomprise (a) the antibody against hepatitis B surface antigen coupled toa water-insoluble, water-insuspensible, solid carrier; (b) said antibodycovalently linked to an enzyme; and (c) hepatitis B surface antigen incase the component to be determined is the antibody against hepatitisantigen.

What is claimed is: .[.
 1. A method for the detection and determinationof a component of an antigen-antibody reaction in a liquid samplecontaining the component to be determined, comprising the steps of:a.providing a given quantity of a first reagent consisting of onecomponent of said reaction selected from the group consisting of anantigen and an antibody bound to the surface of a water-insoluble,water-insuspensible, solid carrier; b. providing a given amount of asecond reagent consisting of a component having the same immunologicalproperties as the component in the said first reagent covalently linkedto an enzyme, and also providing a predetermined amount of the bindingpartner for the component to be determined, when the component in saidliquid sample and the component bound to said solid carrier have thesame immuno-chemical properties; c. contacting a given quantity of saidliquid sample with said reagents forming a reaction mixture having asolid phase and a liquid phase; and d. determining the enzyme activityof either the solid or the liquid phase which is a measure of thepresence and quantity of the component to be determined..]. .[.2. Amethod for determining the presence of a component of anantigen-antibody reaction in a liquid sample containing the component tobe determined comprising the steps of: a. providing a given quantity ofa first reagent consisting of one component of said reaction selectedfrom the group consisting of an antigen and an antibody bound to thesurface of water-insoluble, water-insuspensible, solid carrier, and alsoproviding a predetermined amount of the binding partner for thecomponent to be determined when the component in said liquid sample andthe component bound to said solid carrier have the same immunochemicalproperties; b. contacting and incubating a given quantity of said liquidsample with said first reagent; c. washing said solid carrier; d.providing a given quantity of a substance having the same immunologicalproperties as the component previously bound to the solid carrier, saidsubstance being covalently linked to an enzyme; e. contacting andincubating the solid phase from step (c) with said enzyme-linkedsubstance; f. washing the solid carrer; and g. determining the enzymeactivity substance bound to the solid phase, which is a measure of thepresence and quantity of the component to be determined..]. .[.3. Themethod of claim 2 wherein said liquid sample and the binding partner ofthe component to be determined are preincubated with each other, beforecontacting the sample with said solid carrier..]. .[.4. A method for thedetection and determination of a component in the reaction of ahepatitis antigen and the associated antibody in a liquid samplecontaining said component, comprising the steps of:a. providing a givenquantity of a reagent consisting of one component of said reactionselected from the group consisting of said antigen and said antibodybound to a water-insoluble, water-insuspensible, solid carrier; b.contacting and incubating said liquid sample with said solid carrier ofstep (a) to form a reaction mixture; c. providing a given quantity of asubstance having the same immunological properties as the componentbound to said solid carrier, said substance being covalently linked toan enzyme, and also providing a predetermined amount of the bindingpartner for the component to be determined when the component in saidliquid sample and the component bound to said solid carrier have thesame immunochemical properties; d. contacting and incubating thereaction mixture with said enzyme-linked substance; and p1 e.determining the enzyme activity of substance bound to the solid phase,which is a measure of the presence and quantity of the component to bedetermined..]. .[.5. The method of claim 4 in which said hepatitisantigen is hepatitis B surface antigen, and the antibody is itsassociated antibody..]. .[.6. The method of claim 4 in which said solidcarrier is washed after the incubation of the liquid sample with theimmunological component bound to the solid carrier..]. .[.7. Adiagnostic pack for the detection and determination of a component of anantigen-antibody reaction selected from the group consisting of anantigen and an antibody, consisting of: a. one of said componentscoupled to a water-insoluble, water-insuspensible, solid carrier; b. asubstance having the same immunological properties as the component in(a) covalently linked to an enzyme; and c. the binding partner of thecomponent to be determined if the component to be determined has thesame immunological properties as the component in (a)..]. .[.8. Adiagnostic pack for the detection and determination of hepatitis Bconsisting of: a. the antibody against Hepatitis B surface antigencoupled to a water-insoluble, water-insuspensible, solid carrier; b.said antibody covalently linked to an enzyme; and c. Hepatitis B surfaceantigen in case the component to be determined is the antibody againstHepatitis antigen..]. .Iadd.9. A method for the detection anddetermination of an antibody in a liquid sample containing said antibodyto be detected and to be determined, comprising the steps of:a.providing a given quantity of an antigen to said antibody, said antigenbound to the surface of a water-insoluble, water-insuspensible, solidcarrier; b. contacting and incubating a given quantity of said liquidsample having the antibody to be detected and determined with boundantigen of step (a), forming a reaction mixture having a solid phase anda liquid phase; c. separating the solid phase from the liquid phase; d.contacting and incubating with said solid phase a given quantity of acoupling product obtained by covalently binding a component having thesame immunological properties as said antigen, to an enzyme, whichquantity of coupling product is at least immunochemically equivalent tosaid antigen provided in step (a), in order to form a second solid phaseand a second liquid phase; and e. detecting and determining the enzymeactivity of either the second solid or the second liquid phase of step(d) after separating the solid and liquid phases formed thereby, whichdetection and determination is a measure of the presence and quantity ofsaid antibody to be detected and to be determined..Iaddend. .Iadd.10.The method of claim 9, wherein the quantity of said bound antigen instep (a) is at least sufficient to react with all of the antibody to bedetermined..Iaddend. .Iadd.11. The method of claim 9, wherein anadditional step of separating the solid phase from the liquid phasetakes place between steps (d) and (e)..Iaddend. .Iadd.12. The method ofclaim 11, wherein both separation steps are accomplished by washing thesolid carrier..Iaddend. .Iadd.13. A method for the detection anddetermination of a component of an antigen-antibody reaction in a liquidsample containing said component to be detected and determined,comprising the steps of:a. providing a given quantity of a first reagentconsisting of one component of said reaction selected from the groupconsisting of an antigen and an antibody bound to the surface of awater-insoluble, water-insuspensible, solid carrier with the provisothat said first reagent has the same immunochemical properties as thecomponent to be detected and determined; b. providing a predeterminedamount of a binding partner for said first reagent, which amount is lessthan or is immunochemically equivalent to the given quantity provided instep (a) of the insolubilized component, c. contacting and incubating agiven quantity of said liquid sample having the component to be detectedand determined with said reagents of step (a) and step (b), forming areaction mixture having a solid phase and a liquid phase; d. separatingthe solid phase from the liquid phase; e. contacting and incubating withsaid solid phase a given quantity of a coupling product obtained bycovalently binding a component having the same immunological propertiesas the first reagent, to an enzyme, which quantity of coupling productis at least immunochemically equivalent to the first reagent provided instep (a), in order to form a second solid phase and a second liquidphase; and f. detecting and determining the enzyme activity of eitherthe second solid phase or the second liquid phase of step (e) afterseparating the solid and liquid phases formed thereby, which detectionand determination is a measure of the presence and quantity of thecomponent to be detected and to be determined. .Iaddend. .Iadd.14. Themethod of claim 13, wherein said liquid sample and the binding partnerof the component to be determined are preincubated with each otherbefore contacting the sample with said solid carrier..Iaddend. .Iadd.15.A method for the detection and determination of an antibody in a liquidsample containing the antibody to be detected and determined, comprisingthe steps of:a. providing a given quantity of an antigen to saidantibody, which antigen is bound to the surface of a water-insoluble,water-insuspensible, solid carrier; b. contacting and incubating a givenquantity of said liquid sample having said antibody to be detected andto be determined with said bound antigen of step (a), forming a reactionmixture having a solid phase and a liquid phase; c. washing said solidcarrier to separate the solid phase from the liquid phase; d. contactingand incubating with said solid phase a given quantity of a couplingproduct, obtained by binding covalently a component having the sameimmunological properties as the antigen previously bound to the solidcarrier, to an enzyme, which quantity of coupling product is at leastimmunochemically equivalent to said antigen provided in step (a) to forma second solid and second liquid phase; e. washing the solid carrier toseparate the solid phase from the liquid phase of step (d); and f.detecting and determining the enzyme activity of the solid phase of step(d) after said washing, which detection and determination is a measureof the presence and quantity of said antibody to be detected and to bedetermined..Iaddend. .Iadd.16. The method of claim 15, wherein saidliquid sample and the binding partner of the antibody to be determinedare preincubated with each other, before contacting the sample with saidsolid carrier..Iaddend. .Iadd.17. A method for the detection anddetermination of a component of an antigen-antibody reaction in a liquidsample containing said component to be detected and determined,comprising the steps of:a. providing a given quantity of a first reagentconsisting of one component of said reaction selected from the groupconsisting of an antigen and an antibody, which component is bound tothe surface of a water-insoluble, water-insuspensible, solid carrier,with the proviso that said first reagent has the same immunochemicalproperties as the component to be detected and determined; b. providinga predetermined amount of a binding partner for the first reagent, whichamount is less than or is immunochemically equivalent to the givenquantity provided in step (a) of the insolubilized component, c.contacting and incubating a given quantity of said liquid sample havingthe component to be detected and to be determined with said firstreagent of step (a) and step (b), forming a reaction mixture having asolid phase and a liquid phase; d. washing said solid carrier toseparate the solid phase from the liquid phase; e. contacting andincubating with said solid phase a given quantity of a coupling product,obtained by binding covalently a component having the same immunologicalproperties as the component previously bound to the solid carrier, to anenzyme, which quantity of coupling product is at least immunochemicallyequivalent to the component of the first reagent provided in step (a) toform a second solid and second liquid phase; f. washing the solidcarrier to separate the solid phase from the liquid phase of step (e);and g. detecting and determining the enzyme activity of the solid phaseof step (e) after said washing, which detection and determination is ameasure of the presence and quantity of the component to be detected andto be determined..Iaddend. .Iadd.18. A method for the detection anddetermination of an antibody specific to an hepatitis antigen in aliquid sample containing said antibody, comprising the steps of:a.providing a given quantity of an hepatitis antigen, which antigen isbound to a water-insoluble, water-insuspensible, solid carrier; b.contacting and incubating said liquid sample having the antigen to bedetected and determined with said bound antigen of (a) to form areaction mixture; c. separating the first solid phase from the firstliquid phase; d. contacting and incubating with said solid phase a givenquantity of a coupling product obtained by covalently binding acomponent having the same immunological properties as the antigen boundto said carrier, to an enzyme, which quantity of coupling product is atleast immunochemically equivalent to the first reagent provided in step(a) to form a second solid and second liquid phase; e. contacting andincubating the reaction mixture to form a solid phase and a liquidphase; and f. detecting and determing the enzyme activity of the solidphase of step (d) after separating the solid and liquid phases formedtherein, which detection and determination is a measure of the presenceand quantity of the antibody to be detected and determined..Iaddend..Iadd.19. The method of claim 18 in which said hepatitis antigen ishepatitis B surface antigen and the antibody is its associatedantibody..Iaddend. .Iadd.20. A method of the detection and determinationof a component in the reaction of a hepatitis antigen and the associatedantibody specific to said antigen in a liquid sample containing saidcomponent, comprising the steps of:a. providing a given quantity of afirst reagent consisting of one component of said reaction selected fromthe group consisting of said antigen and said antibody, which componentis bound to a water-insoluble, water-insuspensible, solid carrier withthe proviso that said first reagent has the same immunochemicalproperties as the component to be detected and determined; b. providinga predetermined amount of a binding partner for the first reagent, whichamount is less than or is immunochemically equivalent to the givenquantity provided in step (a) of the insolubilized component; c.contacting and incubating said liquid sample having the component to bedetected and determined with said reagent of step (a) and step (b), toform a reaction mixture; d. separating the first solid phase from thefirst liquid phase; d. contacting and incubating with said solid phase agiven quantity of a coupling product obtained by covalently binding acomponent having the same immunological properties as the componentbound to said carrier, to an enzyme, which quantity of coupling productis at least immunochemically equivalent to the first reagent provided instep (a) to form a second solid and second liquid phase; f. contactingand incubating the reaction mixture to form a solid phase and a liquidphase; and g. detecting and determining the enzyme activity of the solidphase of step (e) after separating the solid and liquid phases formedtherein, which detection and determination is a measure of the presenceand quantity of the component to be detected and determined..Iaddend..Iadd.21. The method of claim 20 in which said hepatitis antigen ishepatitis B surface antigen and the antibody is its associatedantibody..Iaddend. .Iadd.22. A diagnostic pack for the detection anddetermination of a component of an antigen-antibody reaction selectedfrom the group consisting of an antigen and an antibody, consistingof:a. a given quantity of a first reagent consisting of one component ofsaid reaction selected from the group consisting of an antigen and anantibody bound to the surface of a water-insoluble, water-insuspensible,solid carrier; b. a given quantity of a coupling product obtained bycovalently binding a component having the same immunological propertiesas the component in (a) to an enzyme, which quantity of coupling productis at least immunochemically equivalent to the component of the firstreagent provided in (a); and c. a predetermined amount of a bindingpartner of the first reagent, which amount is less than or isimmunochemically equivalent to the given quantity (a) of theinsolubilized component..Iaddend. .Iadd.23. A diagnostic pack for thedetection and determination of an antibody, consisting of:a. a givenquantity of an antigen bound to the surface of a water-insoluble,water-insuspensible, solid carrier; and b. a given quantity of anantigen having the same immunological properties as the antigen in (a)coupled to an enzyme, which quantity of antigen is at leastimmunochemically equivalent to said antigen provided in (a)..Iaddend..Iadd.24. A diagnostic pack for the detection and determination ofantibody directed against hepatitis B, consisting of: a. a givenquantity of hepatitis B surface antigen coupled to a water-insoluble,water-insuspensible, solid carrier; and b. a given quantity of acoupling product obtained by covalently binding hepatitis B surfaceantigen to an enzyme, which quantity is at least immunochemicallyequivalent to the antigen in (a)..Iaddend.